Understanding the mechanism(s) of anticarcinogenic and procarcinogenic effects of beta-carotene is important due to continuing interest in the potential use of carotenoids as chemopreventive agents -- and the conflicting results of observational studies vs. intervention trials. In the last grant period, our studies indicate that a biologic basis for the harmful effect of beta-carotene supplementation in smokers is related to the high dosage used and the enhanced formation of reactive beta-carotene metabolites in the free-radical-rich, but antioxidant-poor environment of the lungs of cigarette smokers. A mechanism to explain the instability of the beta-carotene molecule is that exposure of lung cells to smoke results in increased lung cell oxidative stress, thereby causing decreased tissue levels of other important antioxidants, such as ascorbate and alpha-tocopherol, which have a stabilizing effect on unoxidized beta-carotene. In the present grant, we propose to conduct an intervention to investigate possible protective effects of a rational combination of linked antioxidants (beta-carotene, vitamins C and E) against cigarette smoke-induced lung lesions in the ferret model. These studies will provide important information on the potential future use of rational antioxidant combinations against lung cancer as well as other tissue. Our specific aims are as follows: 1) To determine the effectiveness of beta-carotene in both physiologic (low) and pharmacological (high) doses in the presence of vitamins C and E as anti-proliferative agents in the smoke-exposed ferret by examining A) lung cell proliferation indices (cyclin D1, proliferative cellular nuclear antigen PCNA), and the appearance of squamous metaplasia; B) lung retinoid concentrations and retinoid target gene expressions (RARbeta, MAP kinase phosphatase-1, and Bax-1); C) the Jun N-terminal kinase-dependent (JNK) signaling pathway; and D) apoptosis (Caspase 3 and TUNEL); 2) To determine if there is a dose-dependent relationship between beta-carotene intake in the presence of vitamins C and E and oxidative stress by examining A) beta-carotene, vitamin E and vitamin C concentrations in both plasma and lung tissue; and B) the degree of oxidative stress status by measuring isoprostane and malondialdehyde levels in both plasma and lung tissue of ferret; and 3) To determine if vitamins C and E will inhibit the formation of oxidative metabolites from beta-carotene via in vitro incubation studies using the post-nuclear fraction of ferret lungs. We will also determine whether beta-carotene 9?10?-dioxygenase in the ferret lung is involved in metabolism of beta-carotene into apo-carotenals by examining A) expression of beta-carotene 9?10'?-dioxygenase in the ferret lung tissue after treatment with smoke, pharmacological beta-carotene supplementation, or a combination of both in vivo; and B) whether additional supplementation of vitamins C and E in the smoke-exposed ferret with or without beta-carotene supplementation will regulate the expression of beta-carotene 9?10?-dioxygenase and prevent the destruction of beta-carotene both in vivo and in vitro.